2-Hydroxy-mutilin carbamate derivatives for antibacterial use

ABSTRACT

2-(S)-hydroxymutilin carbamate derivatives of formula (I), in which R 1  is a 5- or 6-membered optionally substituted heteroaryl group; and R 2  is vinyl or ethyl, are useful in the treatment of bacterial infections.

[0001] The present invention relates to novel compounds, to processesfor their preparation, to pharmaceutical compositions containing themand to their use in medical therapy, particularly antibacterial therapy.

[0002] Pleuromutilin, the compound of formula (A), is a naturallyoccurring antibiotic which has antimycoplasmal activity and modestantibacterial activity. Mutilin and other compounds with a free OH atC-14 are inactive. The impact of further modification at C-14 on theactivity of pleuromutilin has been investigated (H. Egger and H.Reinshagen, J. Antibiotics, 1976, 29, 923). Replacing the hydroxy groupof the glycolic ester moiety at position 14 by another O, S or N-linkedgroup was found to improve anti-microbial activity. Thus, introducing adiethylaminoethylthio group gives the compound of formula (B), alsoknown as Tiamulin, which is used as a veterinary antibiotic (G.Hogenauer in Antibiotics, Vol. V, part 1, ed. F. E. Hahn,Springer-Verlag, 1979, p.344).

[0003] In this application, the non-conventional numbering system whichis generally used in the literature (G. Hogenauer, loc. cit.) is used.

[0004] WO 97/25309 (SmithKline Beecham) describes further modificationof the acyloxy group, disclosing inter alia 14-O-acylcarbamoyl(R^(a)CONR^(b)CO₂—) derivatives of mutilin in which R^(a) may have arange of values, including optionally substituted heterocyclic and R^(b)is a selected from a variety of monovalent groups.

[0005] WO 98/05659 (SmithKline Beecham) describes further 14-O-carbamoylderivatives of mutilin in which the N-atom of the carbamoyl group isacylated by a group which includes an azabicyclic moiety.

[0006] WO 99/21855 (SmithKline Beecham) describes further derivatives ofmutilin or 19,20-dihydromutilin, in which the glycolic ester moiety atposition 14 is modified. In such compounds, the 2 position (a to theketogroup) may be substituted by hydroxy. The vast majority of thecompounds exemplified therein, however, do not have such a substituent.

[0007] In addition 19,20-dihydro-2α-hydroxy-mutilin is described by G.Schulz and H. Berner in Tetrahedron, 1984, vol. 40, pp 905-917.

[0008] The present invention is based on the unexpected discovery thatcertain novel 14-O-carbamoyl derivatives mutilin derivatives furtherhaving a (2S)-hydroxy substituent have potent antimicrobial activity.

[0009] Accordingly the present invention provides a compound of formula(I):

[0010] in which:

[0011] R¹ is a 5- or 6-membered optionally substituted heteroaryl group;and

[0012] R² is vinyl or ethyl.

[0013] In this series of compounds, the introduction of a (2S)-hydroxysubstituent is found to impart greater metabolic stability towards liverenzymes than the corresponding 2-unsubstituted counterparts.

[0014] Examples of heteroaryl groups for R¹ include those having a 5 or6-membered single ring comprising 1 or 2 nitrogen atoms and optionallycomprising a further heteroatom selected from oxygen or sulphur, forexample pyridine, pyridazine, pyrimidine, pyrazine, isoxazole, thiazole,imidazole, pyrazole; or a 5 or 6-membered ring comprising 3 nitrogenatoms, for example, 1,2,3-triazole, 1,2,4-triazole; or a 5 or 6-memberedring comprising 1 or 2 nitrogen atoms fused to a benzene ring, forexample, benzimidazole. Further examples of heteroaryl groups for R¹include those having a 5 or 6-membered ring comprising 1 or 2 nitrogenatoms fused to a second 5 or 6-membered optionally substitutedheteroaryl ring comprising 1 or 2 nitrogen atoms.

[0015] Representative examples of such heteroaryl groups for R¹ include,for example, pyridine, pyrazine, pyridazine,3-oxo-3,4-dihydropyrido[2,3-b]pyrazine, pyrazolo[1,5-a]pyrimidine,pyrimidine, and thiazole. Preferred examples of such heteroaryl groupsfor R¹ include, for example, pyridine, pyrimidine, and thiazole.

[0016] Representative optional substituents for R¹ include amino, mono-or di-(C₁₋₆)alkylamino, (C₁₋₆)alkyl, (C₁₋₆)alkoxy, nitro andN-containing heterocyclyl such as piperidin-4-yl which may be optionallysubstituted. Typically R¹ may comprise one or two substituents.

[0017] When used herein, the term “aryl” refers to, unless otherwisedefined, phenyl or naphthyl. A substituted aryl group comprises up tofive, preferably up to three substituents.

[0018] Suitable substituents for an aryl group, including phenyl whenforming part of a benzyl group, include, for example, and unlessotherwise defined, halogen, (C₁₋₆)alkyl, aryl, aryl(C₁₋₆)alkyl,(C₁₋₆)alkoxy, (C₁₋₆)alkoxy(C₁₋₆)alkyl, halo(C₁₋₆)alkyl,aryl(C₁₋₆)alkoxy, hydroxy, nitro, cyano, azido, amino, mono- anddi-N-(C₁₋₆)alkylamino, acylamino, arylcarbonylamino, acyloxy, carboxy,carboxy salts, carboxy esters, carbamoyl, mono- anddi-N-(C₁₋₆)alkylcarbamoyl, (C₁₋₆)alkoxycarbonyl, aryloxycarbonyl,ureido, guanidino, (C₁₋₆)alkylguanidino, amidino, (C₁₋₆)alkylamidino,sulphonylamino, aminosulphonyl, (C₁₋₆)alkylthio, (C₁₋₆)alkylsulphinyl,(C₁₋₆)alkcylsulphonyl, heterocyclyl, heteroaryl, heterocyclyl(C₁₋₆)alkyland heteroaryl(C₁₋₆)alkyl. In addition, two adjacent ring carbon atomsmay be linked by a (C₃₋₅)alkylene chain, to form a carbocyclic ring.

[0019] When used herein, the terms “alkyl” and “alkenyl” refer to(individually or as part of alkoxy or alkenyloxy) straight and branchedgroups containing up to six carbon atoms.

[0020] When used herein, the terms “cycloalkyl” and “cycloalkenyl” referto groups having from three to eight ring carbon atoms.

[0021] When substituted, an alkyl, alkenyl, cycloalkyl or cycloalkenylgroup may comprise up to four substituents, preferably up to twosubstituents. Suitable substituents for alkyl, alkenyl, cycloalkyl orcycloalkenyl groups include aryl, heteroaryl, heterocyclyl,(C₁₋₆)alkoxy, (C₁₋₆)alkylthio, aryl(C₁₋₆)alkoxy, aryl(C₁₋₆)alkylthio,amino, mono- or di-(C₁₋₆)alkylamino, cycloalkyl, cycloalkenyl, carboxyand esters thereof, amide, ureido, guanidino, (C₁₋₆)alkylguanidino,amidino, (C₁₋₆)alkylamidino, (C₁₋₆)acyloxy, azido, hydroxy, and halogen.

[0022] When used herein the terms “heterocyclyl” and “heterocyclic”refer to, unless otherwise defined, non-aromatic, single and fused,rings suitably containing up to four heteroatoms in each ring, each ofwhich is selected from oxygen, nitrogen and sulphur. Each heterocyclicring preferably has from 4 to 7, preferably 5 or 6, ring atoms. A fusedheterocyclic ring system may include carbocyclic rings and need includeonly one heterocyclic ring.

[0023] When substituted, a heterocyclyl group may comprise up to threesubstituents. Preferably a substituent for a heterocyclyl group isselected from oxo, and the group hereinbefore defined as suitable arylsubstituents.

[0024] When used herein, the term “heteroaryl” suitably includes, unlessotherwise defined, a mono- or bicyclic heteroaromatic ring systemcomprising up to four, preferably 1 or 2, heteroatoms each selected fromoxygen, nitrogen and sulphur. Each ring may have from 4 to 7, preferably5 or 6, ring atoms. A bicyclic heteroaromatic ring system may include acarbocyclic ring.

[0025] When substituted, a heteroaryl group may comprise up to threesubstituents. Preferably a substituent for a heteroaryl group isselected from the group hereinbefore defined as suitable arylsubstituents.

[0026] Depending on the substituents, two or more diastereoisomers maybe possible. In that situation the present invention includes theindividual diastereoisomers and mixtures thereof.

[0027] The 2-hydroxy-substituted compounds of formula (I) are of the2-(S) configuration.

[0028] Preferred compounds of the invention include:

[0029] 6-Amino-3-pyridinylcarbonylcarbamic acid 2-(S)-hydroxymutilin14-ester;

[0030] 2-Amino-5-pyrimidinylcarbonylcarbamic acid 2-(S)-hydroxymutilin14-ester;

[0031] 2-Amino-5-thiazolylcarbonylcarbamic acid 2-(S)-hydroxymutilin14-ester; and

[0032] 2-Amino-4-thiazolylcarbonylcarbamic acid 2-(S)-hydroxymutilin14-ester.

[0033] Further preferred compounds include:

[0034] 3-Amino-6-pyridazinylcarbonylcarbamic acid 2-(S)-hydroxymutilin14-ester;

[0035] (2,6-Diamino-4-pyrimidinylcarbonyl)carbamic acid2-(S)hydroxymutilin 14-ester,

[0036] (5-Amino-6-methoxy-3-pyridinylcarbonyl)carbamic acid2-(S)-hydroxymutilin 14-ester;

[0037] (5-Amino-6-methoxy-3-pyridinylcarbonyl)carbamic acid19,20-dihydro-2-(S)hydroxymutilin 14-ester,

[0038] (6-Amino-3-pyridinylcarbonyl)carbamic acid 19,20-dihydro2-(S)-hydroxymutilin 14-ester;

[0039] [2-(1-Piperazinyl)-5-pyrimidinylcarbonyl]carbamic acid2-(S)-hydroxymutilin 14-ester;

[0040] (2-Methylamino-5-pyrimidinylcarbonyl)carbamic acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester;

[0041] (6-Amino-5-methoxy-3-pyridinylcarbonyl)carbamic acid2-(S)-hydroxymutilin 14-ester;

[0042] (6-Dimethylamino-3-pyridinylcarbonyl)carbamic acid2-(S)-hydroxymutilin 14-ester; and

[0043] (6-Methylamino-3-pyridinylcarbonyl)carbamic acid2-(S)-hydroxymutilin 14-ester.

[0044] Particularly preferred compounds include:

[0045] (5-Amino-6-methoxy-3-pyridinylcarbonyl)carbamic acid2-(S)-hydroxymutilin 14-ester;

[0046] (5-Amino-6-methoxy-3-pyridinylcarbonyl)carbamic acid19,20-dihydro-2-(S)hydroxymutilin 14-ester;

[0047] (6-Amino-3-pyridinylcarbonyl)carbamic acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester;

[0048] (6-Dimethylamino-3-pyridinylcarbonyl)carbamic acid2-(S)-hydroxymutilin 14-ester; and

[0049] (3-Amino-6-pyridazinylcarbonyl)carbamic acid 2-(S)-hydroxymutilin14-ester.

[0050] The compounds of this invention may be in crystalline ornon-crystalline form, and, if crystalline, may optionally be hydrated orsolvated. This invention includes within its scope stoichiometrichydrates as well as compounds containing variable amounts of water.

[0051] The compounds according to the invention are suitably provided insubstantially pure form, for example at least 50% pure, suitable atleast 60% pure, advantageously at least 75% pure, preferably at least85% pure, more preferably at least 95% pure, especially at least 98%pure, all percentages being calculated as weight/weight.

[0052] Compounds of the invention that contain a basic group such as anamino substituent may be in the form of a free base or an acid additionsalt. Compounds having an acidic group such as a carboxy substituent maybe in the form of a pharmaceutically acceptable salt. Compounds of theinvention having both a basic and an acidic centre may be in the form ofzwitterions, acid addition salt of the basic centre or alkali metalsalts (of the carboxy group). Pharmaceutically acceptable salts arepreferred.

[0053] Pharmaceutically acceptable acid-addition salts include thosedescribed by Berge, Bighley, and Monkhouse, J. Pharm. Sci., 1977, 66,1-19. Suitable salts include the hydrochloride, maleate, andmethanesulphonate; particularly the hydrochloride.

[0054] Pharmaceutically acceptable salts include those described byBerge, Bighley, and Monkhouse, J. Pharm. Sci., 1977, 66, 1-19. Suitablesalts include alkali metal salts such as the sodium and potassium salts.

[0055] In a further aspect the present invention provides a process forpreparing compounds of formula (I), which process comprises reacting acompound of formula (II):

[0056] (II)

[0057] in which X and P are hydrogen or a hydroxyl protecting group,such as an acyl group, and R² is as hereinbefore defined;

[0058] with an acyl isocyanate of formula R^(1A)CONCO in which R^(1A) isR¹ as hereinbefore defined or a group convertible into R¹, for instancea group comprising a protected substituent therein and thereafter and ifnecessary:

[0059] (a) deprotecting a group X and/or P to generate hydroxyl groupsat position 11 and 2, respectively,

[0060] (b) converting a group R^(1A) to R¹, for instance removing aprotecting group,

[0061] (c) converting a group R¹ to another group R¹, and

[0062] (d) hydrogenating the vinyl group at position 12 to form an ethylgroup.

[0063] Preferably, it is desirable to use a compound of formula (HI) inwhich both P and X are hydroxyl protecting groups.

[0064] Similar such processes have been previously described in WO97/25309 and WO 98/05659 (SmithKline Beecham).

[0065] Methods for preparing acyl isocyanates are described in theliterature. For example, they may be prepared by reaction of an acidchloride (R^(1A)COCl) with silver cyanate (e.g. as described by Murdockand Angier in J. Org. Chem., 1962, 27, 3317), tri-n-butyl tin isocyanate(e.g. as described by Akteries and Jochims, Chem. Ber., 1986, 119, 83),or trimethylsilyl isocyanate (e.g. as described by Sheludyakov et al.,J. Gen. Chem. USSR, 1977, 2061-2067) in an inert solvent such asbenzene, toluene, chloroform, dichloromethane, or 1,2-dichloroethane.Alternatively, they may be prepared by treating a primary amide(R^(1A)CONH₂) or N,N-bis(trimethylsilyl) derivative thereof, with oxalylchloride or phosgene in an inert solvent (e.g. Speziale and Smith, J.Org. Chem., 1962, 27, 3742; Kozyukov, et al., Zh Obshch Khim, 1983, 53,2155).

[0066] The formation and reaction of the acyl isocyanate may beconveniently carried out in one process. This typically involvesreaction of a compound of formula (II) with an acid chloride R^(1A)COClin the presence of silver cyanate and a tertiary base (e.g.triethylamine, diisopropyl ethylamine, pyridine), usually triethylamine,in an inert solvent (e.g. chloroform, dichloromethane,1,2-dichloroethane).

[0067] Thus, in a further aspect the present invention provides aprocess for the preparation of a compound of formula (I) which processcomprises reacting a compound of formula (II) with an acyl chloridecompound of formula R^(1A)COCl, in the presence of silver cyanate and abase, such as triethylamine, and, thereafter, if necessary, carrying outone or more of the following steps in any desired order:

[0068] (e) deprotecting a group P and/or X to generate hydroxyl groupsat position 2 and 11, respectively,

[0069] (f) converting a group R^(1A) to R¹, for instance removing aprotecting group,

[0070] (g) converting one group R¹ to another group R¹, and

[0071] (h) hydrogenating the vinyl group at position 12 to form an ethylgroup.

[0072] Preferably, it is desirable to use a compound of formula (II) inwhich both P and X are hydroxyl protecting groups.

[0073] Suitable hydroxy protecting groups are those well known in theart and which may be removed under conventional conditions and withoutdisrupting the remainder of the molecule. A comprehensive discussion ofthe ways in which hydroxy groups may be protected and methods forcleaving the resulting protected derivatives is given in for example“Protective Groups in Organic Chemistry” (T. W. Greene and P. G. M.Wuts, Wiley-Interscience, New York, 2nd edition, 1991). Particularlysuitable hydroxy protecting groups include, for example, triorganosilylgroups such as, for instance, trialkylsilyl and also organocarbonyl andorganooxycarbonyl groups such as, for instance, acetyl,allyloxycarbonyl, 4-methoxybenzyloxycarbonyl and4-nitrobenzyloxycarbonyl.

[0074] Representative values for P include acetate, dichloroacetate ortrifluoroacetate, preferably dichloroacetate. Representative values forX include acetate, dichloroacetate or trifluoroacetate, preferablytrifluoroacetate. After formation of the 14-O-carbamoyl derivative, the2- and 11-O-acyl groups may be removed by selective hydrolysis (e.g.using NaOH in MeOH).

[0075] Protecting groups which can be used for substituents in R^(1A),for instance amino, carboxy, hydroxy are well known in the art, see forinstance “Protective Groups in Organic Chemistry” (T. W. Greene and P.G. M. Wuts, Wiley-Interscience, New York, 2nd edition, 1991).Particularly suitable hydroxy protecting groups include, for example,triorganosilyl groups such as, for instance, trialkylsilyl and alsoorganocarbonyl and organooxycarbonyl groups such as, for instance,acetyl, allyloxycarbonyl, 4-methoxybenzyloxycarbonyl and4-nitrobenzyloxycarbonyl. Particularly suitable carboxy protectinggroups include alkyl and aryl groups, for instance methyl, ethyl andphenyl. Particularly suitable amino protecting groups includealkoxycarbonyl, 4-methoxybenzyloxycarbonyl and 4-nitrobenzyloxycarbonyl.

[0076] Compounds of formula (I) in which R¹=Et may be prepared byreducing a vinyl group R¹ by hydrogenation over a palladium catalyst(e.g. 10% Palladium-on-carbon) in a solvent such as ethyl acetate,ethanol, dioxane, or tetrahydrofuran, either before or after thecarbamoylation of a compound of formula (II).

[0077] Compounds of formula (II) in which P and X are both hydroxylprotecting groups are novel intermediates which are of use in preparingcompounds of formula (I).

[0078] Accordingly, in a further aspect, the present invention providesfor a compound of formula (II) in which P and X are hydroxyl protectinggroups, in particular an organo-carbonyl group, for instance a(C₁₋₆)alkylcarbonyl group in which the alkyl moiety may be substitutedby from 1 to 3 halogen atoms, for instance trifluoroacetyl anddichloroacetyl. Preferably, P is dichloroacetyl and X istrifluoroacetyl. A preferred compound of formula (II) is:

[0079] (2S)-2-Dichloroacetoxy-11-O-trifluoroacetyl-mutilin.

[0080] A compound of formula (II) may be prepared from mutilin, via anintermediate 2-diazo compound, the preparation of which is similar tothat described by H Berner, et al. in Monatshefte fur Chemie, 1981, vol.112, pp 1441-1450. This intermediate may then be reacted with acarboxylic acid to give a 2-acyloxy-mutilin derivative. Typically,reaction with dichloroacetic acid gives a 2-dichloroacetoxy-mutilinderivative.

[0081] A preferred synthetic route for compounds of formula (I) isoutlined in the following scheme:

[0082] using the following reagents and conditions:

[0083] (i) ethyl formate, sodium methoxide, toluene, room temperature;

[0084] (ii) KOH/EtOH, room temperature;

[0085] (iii) tosyl azide, triethylamine, dichloromethane, −10° C. toroom temperature;

[0086] (iv) dichloroacetic acid, dichloromethane, 0° C. to roomtemperature;

[0087] (v) trifluoroacetyl imidazole, tetrahydrofuran, room temperature;

[0088] (vi) R^(1A)COCl, silver cyanate, triethylamine, dichloromethane,room temperature;

[0089] (vii) 0.5M KOH, EtOH, room temperature.

[0090] The compounds of the present invention may contain a chiralcentre, and therefore the above processes may produce a mixture ofdiastereoisomers. A single diastereoisomer may be prepared by separatingsuch a mixture of diastereoisomers by conventional techniques such aschromatography or fractional crystallisation.

[0091] The compounds of this invention may be in crystalline ornon-crystalline form, and, if crystalline, may optionally be hydrated orsolvated. When some of the compounds of this invention are allowed tocrystallise or are recrystallised from organic solvents, solvent ofcrystallisation may be present in the crystalline product. Similarly,some of the compounds of this invention may be crystallised orrecrystallised from solvents containing water. In such cases water ofhydration may be present in the crystalline product. Crystallisationprocedures will usually produce stoichiometric hydrates. Compoundscontaining variable amounts of water may be produced by processes suchas lyophilisation.

[0092] The compounds according to the invention are suitably provided insubstantially pure form, for example at least 50% pure, suitable atleast 60% pure, advantageously at least 75% pure, preferably at least85% pure, more preferably at least 95% pure, especially at least 98%pure, all percentages being calculated as weight/weight. An impure orless pure form of a compound according to the invention may, forexample, be used in the preparation of a more pure form of the samecompound or of a related compound (for example a correspondingderivative) suitable for pharmaceutical use.

[0093] The present invention also includes pharmaceutically acceptablesalts and derivatives of the compounds of the invention. Salt formationmay be possible when one of the substituents carries an acidic or basicgroup. Salts may be prepared by salt exchange in conventional manner.

[0094] Acid-addition salts may be pharmaceutically acceptable ornon-pharmaceutically acceptable. In the latter case, such salts may beuseful for isolation and purification of the compound of the invention,or intermediates thereto, and will subsequently be converted into apharmaceutically acceptable salt or the free base.

[0095] The compounds of the present invention and their pharmaceuticallyacceptable salts or derivatives have antimicrobial properties and aretherefore of use in therapy, in particular for treating microbialinfections in animals, especially mammals, including humans, inparticular humans and domesticated animals (including farm animals). Thecompounds may be used for the treatment of infections caused by, forexample, Gram-positive and Gram-negative bacteria and mycoplasmas,including, for example, Staphylococcus aureus, Staphylococcusepidermidis, Enterococcus faecalis, Streptococcus pyogenes,Streptococcus agalactiae, Streptococcus pneumoniae, Haemophilus sp.,Neisseria sp., Legionella sp., Chlamydia sp., Moraxella catarrhalis,Mycoplasma pneumoniae, and Mycoplasma gallisepticum.

[0096] The present invention also provides a method of treatingmicrobial infections in animals, especially in humans and indomesticated mammals, which comprises administering a compound of theinvention or a pharmaceutically acceptable salt or derivative or solvatethereof, or a composition according to the invention, to a patient inneed thereof.

[0097] Compounds of the present invention show good activity againstChlamydia pneumoniae. This has been implicated in heart disease, inparticular in promoting vascular infection (see for instance FR 2 771008-A1, Hoechst Marion Roussel SA). Accordingly, in a further aspect,the present invention provides a method of preventing C.pneumoniae—induced atherosclerosis which method comprises treating asubject in need thereof with an effective amount of a compound offormula (I). A compound of formula (I) may also be used in combinationwith an anti-atherosclerotic agent, to reduce the incidence of heartattack and other cardiac events. Representative examples ofanti-atherosclerotic agents include the class of cholesterol-loweringcompounds referred to generically as “statins”, for instanceatorvastatin (Lipitor, Warner Lambert), pravastatin (Pravachol),simvastatin (Lipovas, Merck) and cerivastatin (Baycol, Bayer). It hasalso been suggested that Chlamydia pneumoniae may contribute toAlzheimer's Disease. Accordingly, in a further aspect, the presentinvention provides a method of treating Alzheimer's Disease which methodcomprises treating a subject in need thereof with an effective amount ofa compound of formula (I).

[0098] The invention further provides the use of a compound of theinvention or a pharmaceutically acceptable salt or derivative or solvatethereof in the preparation of a medicament for use in the treatment ofmicrobial infections.

[0099] Compounds of the present invention may be used to treat skin andsoft tissue infections and acne, by topical application. Accordingly, ina further aspect the present invention provides the use of a compound ofthe invention or a pharmaceutically acceptable salt or derivative orsolvate thereof in the preparation of a medicament adapted for topicaladministration for use in the treatment of skin and soft tissueinfections and also in the treatment of acne in humans.

[0100] Compounds of the present invention may be also used for theelimination or reduction of nasal carriage of pathogenic bacteria suchas S. aureus, H. influenzae, S. pneumonia and M. catarrhalis, inparticular colonisation of the nasospharynx by such organisms, by theadministration of a compound of the present invention thereto.Accordingly, in a further aspect, the present invention provides for theuse of a compound of the invention or a pharmaceutically acceptable saltor derivative or solvate thereof in the manufacture of a medicamentadapted for administration to the nasal cavity, for reducing oreliminating the nasal carriage of pathogenic organisms. Preferably, themedicament is adapted for focussed delivery to the nasopharynx, inparticular the anterior nasopharynx.

[0101] Such reduction or elimination of nasal carriage is believed to beuseful in prophylaxis of recurrent acute bacterial sinusitis (RABS) orrecurrent otitis media in humans, in particular in reducing the numberof episodes experienced by a patient over a given period of time orincreasing the time intervals between episodes. Accordingly, in afurther aspect, the present invention provides for the use of a compoundof the invention or a pharmaceutically acceptable salt or derivative orsolvate thereof in the manufacture of a medicament adapted foradministration to the nasal cavity, for prophylaxis of recurrent acutebacterial sinusitis or recurrent otitis media.

[0102] The compounds according to the invention may suitably beadministered to the patient at a daily dosage of from 1.0 to 50 mg/kg ofbody weight. For an adult human (of approximately 70 kg body weight),from 50 to 3000 mg, for example about 1500 mg, of a compound accordingto the invention may be administered daily. Suitably, the dosage foradult humans is from 5 to 20 mg/kg per day. Higher or lower dosages may,however, be used in accordance with normal clinical practice.

[0103] To lessen the risk of encouraging the development of resistantorganisms during prophylaxis of recurrent otitis media or recurrentacute bacterial sinusitis, it is preferred to administer the drug on anintermittent, rather than a continual, basis. In a suitable intermittenttreatment regimen for prophylaxis of recurrent otitis media or recurrentsinusitis, drug substance is administered on a daily basis, for a smallnumber of days, for instance from 2 to 10, suitably 3 to 8, moresuitably about 5 days, the administration then being repeated after aninterval, for instance, on a monthly basis over a period of months, forinstance up to six months. Less preferably, the drug substance may beadministered on a continuing, daily basis, over a prolonged period, forinstance several months. Suitably, for prophylaxis of recurrent otitismedia or recurrent sinusitis, drug substance is administered once ortwice a day. Suitably, drug substance is administered during the wintermonths when bacterial infections such as recurrent otitis media andrecurrent sinusitis tend to be more prevalent. The drug substance may beadministered at a dosage of from 0.05 to 1.00 mg, typically about 0.1 to0.2 mg, in each nostril, once or twice a day.

[0104] More generally, the compounds and compositions according to theinvention may be formulated for administration in any convenient way foruse in human or veterinary medicine, by analogy with other antibiotics.

[0105] Accordingly, in a further aspect, the present invention providesa pharmaceutical composition comprising a compound of the invention or apharmaceutically acceptable salt or derivative or solvate thereoftogether with a pharmaceutically acceptable carrier or excipient.

[0106] The compounds and compositions according to the invention may beformulated for administration by any route, for example oral, topical orparenteral. The compositions may, for example, be made up in the form oftablets, capsules, powders, granules, lozenges, creams, syrups, spraysor liquid preparations, for example solutions or suspensions, which maybe formulated for oral use or in sterile form for parenteraladministration by injection or infusion.

[0107] Tablets and capsules for oral administration may be in unitdosage form, and may contain conventional excipients including, forexample, binding agents, for example, syrup, acacia, gelatin, sorbitol,tragacanth, or polyvinylpyrrolidone; fillers, for example lactose,sugar, maize-starch, calcium phosphate, sorbitol or glycine; tablettinglubricants, for example magnesium stearate, talc, polyethylene glycol orsilica; disintegrants, for example potato starch; and pharmaceuticallyacceptable wetting agents, for example sodium lauryl sulphate. Thetablets may be coated according to methods well known in normalpharmaceutical practice.

[0108] Oral liquid preparations may be in the form of, for example,aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, ormay be presented as a dry product for reconstitution with water oranother suitable vehicle before use. Such liquid preparations maycontain conventional additives, including, for example, suspendingagents, for example sorbitol, methyl cellulose, glucose syrup, gelatin,hydroxyethyl cellulose, carboxymethyl cellulose, aluminium stearate gelor hydrogenated edible fats; emulsifying agents, for example lecithin,sorbitan monooleate or acacia; non-aqueous vehicles (which may includeedible oils), for example almond oil, oily esters (for exampleglycerine), propylene glycol, or ethyl alcohol; preservatives, forexample methyl or propyl p-hydroxybenzoate or sorbic acid; and, ifdesired, conventional flavouring and colour agents.

[0109] Compositions according to the invention intended for topicaladministration may, for example, be in the form of ointments, creams,lotions, eye ointments, eye drops, ear drops, nose drops, nasal sprays,impregnated dressings, and aerosols, and may contain appropriateconventional additives, including, for example, preservatives, solventsto assist drug penetration, and emollients in ointments and creams. Suchtopical formulations may also contain compatible conventional carriers,for example cream or ointment bases, ethanol or oleyl alcohol forlotions and aqueous bases for sprays. Such carriers may constitute fromabout 1% to about 98% by weight of the formulation; more usually theywill constitute up to about 80% by weight of the formulation.

[0110] Compositions according to the invention intended for topicaladministration, in addition to the above, may also contain a steroidalanti-inflammatory agent; for example, betamethasone.

[0111] Compositions according to the invention may be formulated assuppositories, which may contain conventional suppository bases, forexample cocoa-butter or other glycerides.

[0112] Compositions according to the invention intended for parenteraladministration may conveniently be in fluid unit dosage forms, which maybe prepared utilizing the compound and a sterile vehicle, water beingpreferred. The compound, depending on the vehicle and concentrationused, may be either suspended or dissolved in the vehicle. In preparingsolutions, the compound may be dissolved in water for injection andfilter-sterilised before being filled into a suitable vial or ampoule,which is then sealed. Advantageously, conventional additives including,for example, local anaesthetics, preservatives, and buffering agents canbe dissolved in the vehicle. In order to enhance the stability of thesolution, the composition may be frozen after being filled into thevial, and the water removed under vacuum; the resulting dry lyophilisedpowder may then be sealed in the vial and a accompanying vial of waterfor injection may be supplied to reconstitute the liquid prior to use.Parenteral suspensions may be prepared in substantially the same mannerexcept that the compound is suspended in the vehicle instead of beingdissolved and sterilisation cannot be accomplished by filtration. Thecompound may instead be sterilised by exposure to ethylene oxide beforebeing suspended in the sterile vehicle. Advantageously, a surfactant orwetting agent is included in such suspensions in order to facilitateuniform distribution of the compound.

[0113] A compound or composition according to the invention is suitablyadministered to the patient in an antimicrobially effective amount.

[0114] A composition according to the invention may suitably containfrom 0.001% by weight, preferably (for other than spray compositions)from 10 to 60% by weight, of a compound according to the invention(based on the total weight of the composition), depending on the methodof administration.

[0115] When the compositions according to the invention are presented inunit dosage form, for instance as a tablet, each unit dose may suitablycomprise from 25 to 1000 mg, preferable from 50 to 500 mg, of a compoundaccording to the invention.

[0116] Representative compositions of the present invention includethose adapted for intranasal administration, in particular, those thatwill reach into the nasopharynx. Such compositions are preferablyadapted for focussed delivery to, and residence within, the nasopharynx.The term ‘focussed delivery’ is used to mean that the composition isdelivered to the nasopharynx, rather than remaining within the nares.The term ‘residence’ within the nasopharynx is used to mean that thecomposition, once delivered to the nasopharynx, remains within thenasopharynx over a course of several hours, rather than being washedaway more or less immediately. Preferred compositions include spraycompositions and creams. Representative spray compositions includeaqueous compositions, as well as oily compositions that containamphiphilic agents so that the composition increases in viscosity whenin contact with moisture. Creams may also be used, especially creamshaving a rheology that allows the cream to spread readily in thenasopharynx.

[0117] Preferred aqueous spray compositions include, in addition towater, further excipients including a tonicity modifier such as a salt,for instance sodium chloride; preservative, such as benzalkonium salt; asurfactant such as a non-ionic surfactant, for instance a polysorbate;and buffer, such as sodium dihydrogen phosphate; present in low levels,typically less than 1%.

[0118] The pH of the composition may also be adjusted, for optimumstability of the drug substance during storage. For compounds of thepresent invention, a pH in the range 5 to 6, preferably about 5.3 to5.8, typically about 5.5 is optimal.

[0119] Representative oily spray and cream compositions are described inWO 99/07341 and WO 99/12520 (SmithKline Beecham). Representative aqueoussprays have previously been described in WO 99/21855 (SmithKlineBeecham).

[0120] Suitably, the drug substance is present in compositions for nasaldelivery in between 0.001 and 5%, preferably 0.005 and 3%, by weight ofthe composition. Suitable amounts include 0.5% and 1% by weight of thecomposition (for oily compositions and creams) and from 0.01 to 0.2%(aqueous compositions).

[0121] Spray compositions according to the present invention may bedelivered to the nasal cavity by spray devices well known in the art fornasal sprays, for instance an air lift pump. Preferred devices includethose that are metered to provide a unit volume of composition,preferably about 100 μl, and optionally adapted for nasal administrationby addition of a modified nozzle.

[0122] The invention is illustrated by the following Examples.

[0123] Note on Naming of Pleuromutilin Analogues

[0124] The compound of formula (a) has, under the IUPAC system, thesystematic name (1S, 2R, 3S, 4S, 6R, 7R, 8R,14R)-3,6-dihydroxy-2,4,7,14-tetramethyl4-vinyl-tricyclo[5.4.3.0^(1,8)]tetradecan-9-one.It is also referred to using the trivial name mutilin and with thenumbering system described by H. Bemer, G. Schulz, and H. Schneider inTetrahedron, 1981, 37, 915-919.

[0125] Preparation 1 (2S)-2-Dichloroacetoxy-11-O-trifluoroacetyl-mutilin

[0126] (a) Formylated derivatives of mutilin The reaction was carriedout similarly to that described by A. J. Birch, C. W. Holzapfel and R.W. Rickards (Tet (Suppl) 1996 8 part III 359). Mutilin (6 g) in toluene(330 ml) and methyl formate (100 ml) was treated with sodium methoxide(3 g) and stirred under argon for 8 hours. Ice-water (100 ml) was added,followed by 2N HCl (220 ml). The mixture was shaken and separated andthe aqueous extracted with ether. The combined organic was dried andevaporated and the residue chromatographed, eluting with ethylacetate/hexane mixtures. First eluted was 2-hydroxymethylenemutilin11,14-diformate (2.33 g): ¹HNMR (CDCl₃) inter alia 5.02 (1H, d), 5.77(1H, d), 6.94 (1H, s), 7.89 (1H, s), 8.10 (1H, s). Second to be elutedwas 2-hydroxymethylenemutilin 11-formate (3.0 g): ¹H NMR (CDCl₃) interalia 4.40 (1H, d), 5.11 (1H, d), 7.06 (1H, s), 8.25 (1H, d, J O.8 Hz).Third to be eluted was a mixture (2:1) of 2-hydroxymethylenemutilin14-formate and 2-hydroxymethylenemutilin (1.8 g).

[0127] (b) 2-Hydroxymethylenemutilin A mixture of2-hydroxymethylenemutilin 11,14-diformate (2.33 g) and[2-hydroxymethylenemutilin 14-formate+2-hydroxymethylene mutilin] (1.8g) was dissolved in ethanol (30 ml) and treated with 0.5M KOH in ethanol(60 ml). After 1 hour the solution was diluted with ethyl acetate (200ml), washed with 2M HCl (120 ml) and water (100 ml), dried andevaporated to provide 2-hydroxymethylenemutilin as a foam (3.6 g); ¹HNMR (CDCl₃) inter alia 3.45 (1H, d), 4.37 (1H, d), 6.97 (1H, s).

[0128] (c) 2-Diazomutilin A solution of 2-hydroxymethylenemutilin (3.6g) in dichloromethane was cooled to −10° C. under argon, treated withtriethylamine (4.6 ml) and tosyl azide (3.55 g) and warmed to roomtemperature. After 6 hours the solution was washed with 0.5M HCl(150 ml)and water (100 ml), dried and evaporated. The 2-diazomutilin wasobtained as yellow crystals (1.7 g) from ethyl acetate/hexane; IR(CHCl₃) 3634, 2082 and 1670 cm⁻¹.

[0129] (d) (2S)-2-Dichloroacetoxymutilin A solution of 2-diazomutilin(1.7 g) in dichloromethane (40 ml) was ice-cooled and treated dropwisewith dichloracetic acid (0.5 ml). The bath was removed and after 30minutes the solution was colourless. It was washed with aqueous NaHCO₃(50 ml), dried and evaporated. Chromatography, eluting with 1:3 ethylacetate/hexane, gave the title compound as the less polar of 2 majorproducts (white foam, 1.6 g): ¹H NMR (CDCl₃) inter alia 3.33 (1H, t, J5.8 Hz), 4.33 (1H, d, J 7 Hz), 5.04 (1H, t, J 9 Hz), 5.2-5.4 (2H, m),5.96 (1H, s), 6.14 (1H, dd, J 17.5 and 10.5 Hz).

[0130] (e) (2S)-2-Dichloroacetoxy-11-O-trifluoroacetylmutilin(2S)-2-Dichloroacetoxymutilin (5.8 g, 0.012 mole) in dry tetrahydrofuran(120 ml) was treated with trifluoroacetylimidazole (1.54 ml, 0.0135mole) and stirred at ambient temperature for 18 hours. Ethyl acetate(200 ml) was added to the mixture which was then washed with dilutesodium chloride solution (2×200 ml). The organic layer was separated,dried (Na₂SO₄), filtered and evaporated to dryness. Chromatography onsilica gel, eluting with ethyl acetate/hexane (9:1) gave the titlecompound (4.98 g, 71%); ¹H NMR (CDCl₃) inter alia 0.85 (3H, d, J 7 Hz),0.95 (3H, d, J 7 Hz), 1.05 (3H, s), 1.39 (3H, s), 4.29 (1H, t, J 7 Hz),4.86 (1H, d, J 7 Hz), 5.08 (1H, t, J 9 Hz), 5.99 (1H, s).

[0131] Preparation 2 6-tert-Butyloxycarbonylaminonicotinic Acid

[0132] Methyl 6-aminonicotinate (log) in t-butanol (500 ml) was treatedwith di-tert-butyldicarbonate (15.8 g) and heated at 100° C. for 36hours. The mixture was concentrated in-vacuo. Trituration with diethylether gave methyl 6-tert-butyloxycarbonylaminonicotinate (12.8 g).Treatment of this compound with lithium hydroxide monohydrate in amixture of tetrahydrofuiran (150 ml) and water (150 ml) for 18 hours andevaporating to a small volume was followed by acidification with citricacid. Filtration gave the title compound as a white solid (8.99 g, 57%).M.S.(−ve ion chemical ionisation)m/z 237 ([M-H]⁻, 80%), 193 (100%).

[0133] Preparation 3 6-tert-Butyloxycarbonylaminoisonicotinic Acid

[0134] The title compound was prepared analogously to Preparation 2 frommethyl 6-aminoisonicotinate (D. J. Stanonis, J. Org. Chem. 22 (1957)475)to give 1.54 g. M.S.(−ve ion chemical ionisation)m/z 237 ([M-H]⁻, 55%),193 (100%).

[0135] Preparation 4 Sodium5-bis-t-butoxyearbonylaminopyridin-3-ylcarboxylate

[0136] (a) Ethyl 5-aminonicotinate 5-Aminonicotinic acid (2.2 g)(Bachman and Micucci, J. Amer. Chem. Soc. 70 (1948) 2381) in ethanol (20ml) was ice-cooled, saturated with HCl gas and refluxed 4 hours. Themixture was concentrated to low volume and partitioned between EtOAc(100 ml) and saturated NaHCO₃ solution (100 ml). The organic phase waswashed with further aqueous NaHCO₃, dried and evaporated to leave thetitle compound as a white solid (1.34 g). M.S. (+ve ion chemicalionisation) m/z 167 (MH⁺,100%).

[0137] (b) Ethyl 5-bis-t-butoxycarbonylaminopyridin-3-yl carboxylate Asolution of ethyl 5-aminonicotinate (1.3 g) in 1,2-dichloroethane (20ml) was treated with triethylamine (2.4 ml), di-t-butyldicarbonate (5.12g) and 4-dimethylaminopyridine (14 mg) and refluxed 1 hour. The solventwas evaporated and the residue taken up in EtOAc (50 ml), washed withwater (2×50 ml), dried and evaporated. Chromatography gave the titlecompound as a white solid (947 mg). M.S. (+ve ion chemical ionisation)m/z 367 (MH⁺, 40%), 167 (100%).

[0138] (c) Sodium 5-bis-t-butoxycarbonylaminopyridin-3-ylcarboxylate Asolution of ethyl 5-bis-t-butoxycarbonylaminopyridin-3-ylcarboxylate(0.9 g) in dioxan (15 ml)/water (1 ml) was treated with 2N aqueous NaOH(1.62 ml) and stirred overnight. The solution was evaporated to give thetitle compound as a solid, which was dried under vacuum (0.912 g). M.S.(+ve ion chemical ionisation) m/z 339 (MH⁺ free acid, 3%), 167 (100%).

[0139] Preparation 5 Sodium6-bis-t-butoxycarbonylaminopyridin-2-ylcarboxylate The title compoundwas prepared analogously to Preparation 4, steps 2 and 3 from ethyl6-aminopyridin-2 ylcarboxylate (Ferrari and Marcon, Farmaco Ed. Sci. 14(1959) 594-596) in quantitative overall yield. NMR δ (CD₃OD) 1.39 (18H,s), 7.33 (1H, dd), 7.76 (1H, t), 7.95 (1H, dd).

[0140] Preparation 6 Sodium5-bis-t-butoxycarbonylaminopyridin-2-ylcarboxylate The title compoundwas prepared analogously to Preparation 4, steps 2 and 3 from methyl5-aminopyridin-2-ylcarboxylate (O. P. Shkurko and V. P. Mamaev, Chem.Heterocycl. Compd. 26 (1990)47-52) in 52% overall yield. NMR δ (D₂O)1.35 (18H, s), 7.77 (1H, dd), 7.92 (1H, d), 8.38 (1H, d).

[0141] Preparation 7 Sodium4-bis-t-butoxycarbonylaminopyridin-2-ylcarboxylate (a) Methyl4-aminopyridin-2-ylcarboxylate A solution of methyl4-nitropyridin-2-ylcarboxylate (0.7 g) (Deady et. al., Aus. J. Chem. 24(1971)385-390) in methanol (30 ml) was treated with 10% Pd/C (0.3 g) andstirred under hydrogen at atmospheric pressure overnight. The solutionwas filtered and evaporated to yield the title compound (0.55 g). NMR δ(CDCl₃) 3.97 (3H, s), 4.34 (2H, broad), 6.65 (1H, dd), 7.39 (1H, d),8.32 (1H, d).

[0142] (b) and (c) were carried out analogously to steps (b) and (c) ofpreparation 4 to provide the title sodium salt in overall 67% yield.MS(−ve ion chemical ionisation) m/z 337 ([M-H]⁻ free acid, 70%, 178(100%).

[0143] Preparation 8 Sodium 6-methoxynicotinate Hydrolysis of methyl6-methoxynicotinate in a manner analogous to step (c) of preparation 4provided the title compound.

[0144] Preparation 9 2-t-butoxycarbonylaminothiazole-5-carboxylic Acid

[0145] (a) Methyl 2-bis-t-butoxycarbonylaminothiazole-5-carboxylate Asolution of methyl 2-aminothiazole-5-carboxylate (2.3 g) (R. Noto, M.Ciofalo, F. Buccheri, G. Werber and D. Spinelli, J C S Perkin Trans. 2,(1991)349-352) in dichloromethane (60 ml) was treated with triethylamine(2 ml), a catalytic amount of 4-dimethylaminopyridine anddi-t-butyldicarbonate (8 g) and stirred overnight. The solution wasevaporated to low volume, applied to a silica column and eluted withethyl acetate/hexane to provide the title compound (3.56 g).

[0146] (b) 2-t-Butoxycarbonylaminothiazole-5-carboxylic acid A solutionof methyl 2-bis-t-butoxycarbonylaminothiazole-5-carboxylate (3.56 g) indioxan (50 ml) was treated with 2N NaOH solution (9 ml), stirred 1 hour,treated with a further 17 ml of 2N NaOH and stirred a further hour. Themixture was taken to pH 8 with 2N HCl and evaporated. The solid wastaken up in water (10 ml), treated with a solution of citric acid (6.6g) in water (20 ml) and extracted with ethyl acetate (30 ml). The ethylacetate was separated, washed with water (3×20 ml), dried and evaporatedto yield the title compound as a solid (0.96 g). NMR δ (DMSO) 1.50 (9H,s), 7.95 (1H, s), 11.90 (1H, broad).

[0147] Preparation 10 2-t-Butoxycarbonylaminothiazole-4-carboxylic Acid

[0148] (a) Ethyl 2-aminothiazole-4-carboxylate2-Aminothiazole-4-carboxylic acid hydrobromide (10 g) (E. C. Roberts andY. F. Shealy, J. Med. Chem. 15 (1972)1310-1312) in ethanol (35 ml) wastreated with conc. sulfuric acid and refluxed for 48 hours. The solutionwas evaporated to 25% of original volume and water (20 ml) added. It wasmade basic by addition of NaHCO₃, the solid filtered, washed with waterand dried under vacuum to give the title compound (5.64 g). NMR δ(CDCl₃) 137 (3H, t), 4.36 (2H, q), 5.39 (2H, broad), 7.43 (1H, s).

[0149] (b) and (c) were carried out analogously to steps (b) and (c) ofpreparation 9 to provide the title acid. NMR (CD₃OD) 1.45 (9H, s), 7.77(1H, s).

[0150] Preparation 11 Sodium 2,6-bis(bis-t-butoxycarbonylamino)pyrimidine-4-carboxylate

[0151] (a) Methyl 2,6-diaminopyrimidine-4-carboxylate 2,6-diaminopyrimidine-4-carboxylic acid (G. D. Davies, F. Baiocchi, R. K. Robinsand C. C. Cheng, J. Org Chem 26 (1961) 2755-2759) was esterified withHCl/MeOH using the procedure of Preparation 4, step (a) in 100% yield.¹HNMR δ (DMSO) 3.90 (3H, s), 6.72 (1H, s), 8.57 (broad), 8.93 (broad).

[0152] (b) was carried out analogously to step (a) of Preparation 9 and(c) analogously to step (c) of Preparation 4 to give the title compound(30% over 2 steps). ¹HNMR δ (DMSO) 1.38 (18H, s), 4.45 (18H, s), 7.71(1H, s).

[0153] Preparation 122-(1-t-butoxycarbonylpiperidin-4-yl)thiazole-4-carboxylic Acid

[0154] A solution of ethyl 2-(1-t-butoxycarbonylpiperidin-4-yl)thiazole-4-carboxylate (from Tripos UK Ltd) (340 mg) in dioxan (5ml)/water (1 ml) was treated with 2N NaOH (0.6 ml) and left overnight.The solution was diluted with EtOAc (20 ml) and 1M citric acid solution(10 ml), shaken, separated. The organic was washed with water (3×10 ml),dried and evaporated to give the title compound as a solid (295 mg). MS(+ve ion electrospray) m/z 335 (MNa⁺, 30%) 239 (100%);(−ve ionelectrospray) m/z 267 ([M-COOH]⁻, 100%).

[0155] Preparation 13 2-Methoxypyrimidine-5-carboxylic acid A solutionof methyl 2-methoxypyrimidine-5-carboxylate (944 mg) (Z. Budesinsky andJ. Vavrina, Collect. Czech. Chem. Commun. 37 (1972)1721-1733) in dioxan(33 ml)/water (33 ml) was treated with 2N NaOH (3.37 ml), left overnightand evaporated to low volume. The residue was taken up in water (30 ml),the pH adjusted to 2 by addition of 2N HCl and the mixture extractedwith EtOAc (4×30 ml). The EtOAc was dried and evaporated to give thetitle compound as a white solid (605 mg) ¹HNMR δ (DMSO) 4.00 (3H, s),9.03 (2H, s).

[0156] Preparation 14(2S)-2-Dichloroacetoxy-19,20-dihydro-11-O-trifluoroacetylmutilin2-Diazo-19,20, dihydromutilin(H. Bemer, G. Schulz and G. Fischer,Monatsh. für Chemie, 112 (1981)1441-1450) was treated as in Preparation1 steps (d) and (e) to provide the title compound. MS (−ve ionelectrospray)m/z 603 (MOAc⁻, 65%), 543 ([M-H]⁻, 100%).

[0157] Preparation 15 Sodium2-bis-t-butoxycarbonylaminopyrazine-5-carboxylate

[0158] Ethyl 2-aminopyrazine-5-carboxylate (E. Felder, D. Pitre and E.B. Grabitz, Helv. Chim. Acta 47 (1964) 873-876) was treated analogouslyto step (b) of Preparation 9 and then step (c) of Preparation 4 to givethe title compound as a white solid. NMR δ (DMSO) 1.38 (18H, s), 8.51(1H, s), 8.88 (1H, s)

[0159] Preparation 16 Sodium2-N-t-butoxycarbonyl-N-methylaminopyrimidine-5-carboxylate

[0160] 2-N-methylaminopyrimidine-5-carboxylic acid (D. J. Brown and M.N. Paddon-Row, J. Chem. Soc. C, (1966) 164-166) was esterified using theprocedure of Preparation 4 (step (a). The ester was treated according tostep (a) of Preparation 9 and then step (c) of Preparation 4 to give thetitle compound. NMR δ (DMSO) 1.42 (9H, s), 3.28 (3H, s) and 8.91 (2H,s).

[0161] Preparation 17 Sodium5-bis-t-butoxycarbonylamino-6-methoxynicotinate

[0162] Methyl 5-amino-6-methoxynicotinate (Morisawa et. al., Agric.Biol. Chem. 40, (1976) 101) was treated according to step (a) ofPreparation 9 and then step (c) of Preparation 4 to give the titlecompound. MS (−ve ion chemical ionisation) m/z 367 ([M-H]⁻, 100%).

[0163] Preparation 18 Sodium6-bis-t-butoxycarbonylamino-5-methoxynicotinate

[0164] (a) Methyl 6-amino-5-methoxynicotinate A mixture of2-amino-5-bromo-3-methoxypyridine (7 g) (den Hertog et al, Recl. Trav.Chim. Pays-Bas, 74 (1955), 1171), bis(triphenylphosphine)palladiumdibromide (3.5 g) and tri-n-butylamine (9 ml) in methanol (35 ml) wassubjected to 80 psi pressure of carbon monoxide and heated at 112° C.for 16 hours. The mixture was cooled and evaporated and the residuechromatographed, eluting with 1:1 EtOAc/hexane to give the titlecompound (2.32 g). MS (+ve ion chemical ionisation) m/z 183 (MH⁺, 100%).

[0165] (b) and (c) were carried out analogously to Preparation 9, step(a) and Preparation 4, step (c) to give sodium6-bis-t-butoxycarbonylamino-5-methoxynicotinate (overall 77%). MS (−veion chemical ionisation) m/z 367 ([M-H]⁻, 100%).

[0166] Preparation 19 Sodium6-bis-t-butoxycarbonylamino-5-nitronicotinate

[0167] 6-Amino-5-nitronicotinic acid (Marckwald, Chem. Ber. 27, (1894),1336) was esterified by the procedure of Preparation 4, step (a),N-protected as described in Preparation 9, step (a) and the esterhydrolysed by the procedure of Preparation 4, step (c) to give the titlecompound. NMR δ (DMSO) 1.32 (18H, s), 8.72 (1H, s), 9.07 (1H, s)

[0168] Preparation 20 Sodium2-bis-t-butoxycarbonylamino-6-methoxypyrimidine-4-carboxylate

[0169] (a) Methyl 2-chloro-6-methoxypyrimidine-4-carboxylate Methyl2,6-dichloropyrimidine-4-carboxylate (10 g) (M. Winn et. al., J. Med.Chem. 36 (18), (1993), 2676-2688) in methanol (100 ml) was treated withsodium ethoxide (3 g) and left for 16 hours. Methanol was evaporated andthe residue partitioned between dichloromethane and saturated aqueousNaHCO₃. The organic was washed with brine, dried and evaporated to givethe title compound (24%). NMR δ (CDCl₃) 4.00 (3H, s), 4.07 (3H, s), 7.37(1H, s).

[0170] (b) Sodium 2-chloro-6-methoxypyrimidine-4-carboxylate Methylester (a) was hydrolysed according to preparation 4, step (c), to givetitle compound (100%). NMR δ (DMSO) 3.93 (3H, s), 7.04 (1H, s)

[0171] (c) Methyl 2-amino-6-methoxypyrimidine 4- carboxylate A solutionof sodium 2-chloro-6-methoxypyrimidine-4-carboxylate (2 g) in conc.aqueous ammonia (30 ml) was refluxed 4 hours and evaporated to dryness.The residue was taken up in methanol (200 ml) treated with conc.sulfuric acid (1 ml) and refluxed 16 hours. After evaporation to lowvolume, the mixture was partitioned between EtOAc and saturated aqueousNaHCO₃. The organic was washed with brine, dried and evaporated to givethe title compound as a white solid (700 mg). NMR δ (CD₃OD) 3.92 (3H,s), 3.94 (3H, s), 6.81 (1H, s).

[0172] (d) Sodium2-bis-t-butoxycarbonylaminio-6-methoxypryimidine-4-carboxylateAminopyrimidine (c) was protected according to the procedure ofPreparation 4, step (b) and the ester hydrolysed according to theprocedure of Preparation 4, step (c) to give the title compound.

[0173] Preparation 21 Sodium2-bis-t-butoxycarbonylaminopyrinmidin-4-ylcarboxylate

[0174] The title compound was prepared analogously to Preparation 4 from2-aminopyrimidine-4-carboxylic acid (T. Matsukawa, K. Shirakawa, J.Pharm. Soc. Japan (1952), 72, 909-912). NMR δ (DMSO) 1.39 (18H, s), 7.59(1H, d, J 5 Hz), 8.72 (1H, d, J 5 Hz)

[0175] Preparation 22 6-N-t-Butoxycarbonyl-N-methylaminonicotinic Acid

[0176] (a) 6-Methylaminonicotinic acid hydrochloride 6-chloronicotinicacid (4.5 g) was dissolved in methanol (50 ml), treated with 33%methylamine in ethanol solution (25 ml) and heated in a sealed bomb at140° C. for 18 hours. The mixture was cooled and evaporated to dryness.Trituration with 1:1 methanol/diethyl ether gave the title compound (3.7g, 69%). MS (+ve is an electrospray) m/z 153 (MH⁺, 100%).

[0177] (b) Methyl (6-methylaminonicotinate 6-Methylaminonicotinic acidhydrochloride (3.65 g) in methanol (100 ml) was treated with conc.sulphuric acid (2 ml) and heated under reflux for 18 hours. The mixturewas evaporated to dryness and the residue partitioned between ethylacetate and saturated sodium bicarbonate solution. The organic layer wasdried and evaporated to dryness to give the title compound (1.07 g) M.S(+ve ion electrospray) m/z 167 (MH⁺, 100%)

[0178] (c) Methyl 6N-t-butoxycarbonyl-N-methylamino nicotinate The titlecompound was prepared analogously to preparation 4, step (b) to give(1.41 g, 58%)

[0179] (d) 6-N-t-Butoxycarbonyl-N-methylaminonicotinic Acid

[0180] Ester hydrolysis was carried out analogously to the esterhydrolysis in Preparation 2 to give the title compound (76%). MS (−veion chemical ionisation) m/z 251 ([M-H]⁻, 100%)

[0181] Preparation 23 Sodium 3-(N-t-butoxycarbonyl-N-methylamino)pyridazine-6-carboxylate

[0182] (a) 3-Methylaminopyridazine-carboxylic acid3-Chloropyridazine-6-carboxylic acid (2.5 g) (R. F. Homer, H. Gregory,W. G. Overend and L. F. Wiggins, J. Chem. Soc (1948) 2195-9) was treatedwith 8 M methylamine in ethanol (2.16 ml) and heated at 100° C. in asealed bomb for 18 hours. The solution was acidified to pH 4 with 5N HCland the precipitate filtered off to provide title compound (0.58 g). MS(−ve ion chemical ionisation) ml/z 152 ([M-H]⁻, 100%).

[0183] (b) Ethyl 3-methylaminopyridazine-6-carboxylate A solution of3-methylaminopyridazine-6-carboxylic acid (0.58 g) in ethanol (50 ml)was saturated with HCl gas, refluxed 48 hours and evaporated. Theresidue was partitioned between EtOAc and aqueous NaHCO₃, separated andthe aqueous re-extracted with EtOAc. The organic was dried andevaporated to give title compound (0.61 g). MS(+ve ion chemicalionisation) m/z 182 (MH⁺, 100%).

[0184] (c) Ethyl3-(N-t-butoxycarbonyl-N-methylamino)pyridazine-6-carboxylate Preparationanalogous to Preparation 9, step (a) (72%). MS (+ve ion chemicalionisation) m/z 282 (MH⁺, 100%).

[0185] (d) Sodium3-(N-t-butoxycarbonyl-N-methylamino)pyridazine-6-carboxylate Preparationanalogous to Preparation 4, step (c) (93%). MS (−ve ion chemicalionisation) m/z 252 ([M-H]⁻, 100%)

[0186] Preparation 24 Sodium6-(bis-t-butoxycarbonylamino)-5-cyanonicotinate

[0187] (a) 6-Hydroxy-5-iodonicotinic acid 6-Hydroxynicotinic acid (20 g)in water (200 ml) and H₂SO₄ (80 ml) was heated to 90° C. for 1 hour.Potassium iodate (0.42 equivalent) and potassium iodide (0.96equivalent) were both added portionwise over 2 hours. After a furtherhour at 90° C. the mixture was cooled to 60° C. and added to 1 kg ofice. The brown solid was filtered off, dried and taken up in DMF (30ml)/EtOH(1 litre). Sodium metabisulfite was added until the brown colourdisappeared and the mixture was poured onto ice (2 kg), a further 1.5litre water added and the white solid filtered to give title compound(16.5 g). NMR δ (DMSO) 12.95 (1H, broad), 12.35 (1H, broad), 8.36 (1H,d), 8.03 (1H, d)

[0188] (b) Methyl 6-chloro-5-iodonicotinate 6-Hydroxy-5-iodonicotinicacid (15.25 g) was refluxed 4 hours in thionyl chloride (40 ml)/DMF (5ml), cooled and evaporated to dryness. The residue was taken up inchloroform (50 ml) and added to methanol (100 ml). Evaporation gave thetitle compound (17 g). NMR δ (CDCl₃) 8.92 (1H, d), 8.71 (1H, d), 3.96(3H, s).

[0189] (c) Sodium 6-chloro-5-iodonicotinate Preparation analogous toPreparation 4, step (c) (100%). NMR δ (DMSO) 8.72 (1H, d), 8.59 (1H, d).

[0190] (d) Methyl 6-amino-5-iodonicotinate Sodium6-chloro-5-iodonicotinate (5 g) in 0.88 ammonia solution (125 ml) washeated at 150° C. for 18 hours in a sealed bomb, cooled and evaporatedto dryness. The residue was esterified according to the procedure ofPreparation 22 step (b) (2.44 g). MS (−ve ion chemical ionisation) m/z277 ([M-H]⁻, 100%).

[0191] (e) Methyl 6-amino-5-cyanonicotinate A mixture of methyl6-amino-5-iodonicotinate (2.44 g), tris(dibenzylideneacetone)dipalladium (0) (4% by weight), 1,1′-bis(diphenylphosphino)ferrocene(16% by weight) and cuprous cyanide (4 equivalents) in dioxan (50 ml)was refluxed for 4 hours, cooled and filtered. The filtrate wasevaporated and the residue chromatographed, eluting with 4% MeOH/CH₂Cl₂to give title compound (1.45 g). NMR δ (DMSO) 8.95 (1H, d), 8.69 (1H,d), 7.79 (2H, broad), 3.80 (3H, s).

[0192] (f) Methyl 6-(bis-t-butoxycarbonylamino)-5-cyanonicotinatePreparation analogous to Preparation 9, step (a) (73%). NMR δ (CDCl₃)9.25 (1H, d), 8.60 (1H, d), 4.01 (3H, s), 1.46 (18H, s).

[0193] (g) Sodium 6-(bis-t-butoxycarbonylamino)-5-cyanonicotinatePreparation analogous to Preparation 4, step (c) (100%). NMR δ (D₂O)9.03 (1H, d), 8.06 (1H, d), 1.32 (18H, s).

[0194] Pyrimidine-5-carboxylic acid was prepared according to I. T.Forbes, R. T. Martin and G. E. Jones, Preparation of indolylureaderivatives as antagonists, PCT Int. Appl. (1993) WO9318028 A1 19930916.

[0195]2-Dimethylaminopyrimidine-5-carboxylic acid was prepared accordingto P. Dorigo, D. Fraccarollo, G. Santostasi, I. Maragno and M. Floreani,J. Med. Chem. 39 (1996) 3671-3683.

[0196] Pyrazolo [1,5-a] pyrimidine-3-carboxylic acid was obtained fromChembridge.

[0197] 6-Dimethylaminonicotinic acid was preapred according toTschitschibabin et. al., Chem. Ber. (1929), 62, 3052.

[0198] 3-Chloropyridazine-6-carboxylic acid was prepared according toR.F. Homer, H. Gregory, W. G. Overend and L. F. Wiggins, J. Chem. Soc.(1948), 2195-2199.

EXAMPLE 1 6-Amino-3-pyridinylcarbonyl)carbamic acid 2-(S)-hydroxymutilin14-ester

[0199]

[0200] (a) (6-tert-Butyloxcarbonylamino-3-pyridinylcarbonyl)carbamicAcid-2-(S)2-dichloroacetoxymutilin 14-ester-11-trifluoroacetate

[0201] 6-tert-Butyloxycarbonylaminonicotinic acid (1.0 g) indichloromethane (100 ml) was treated with oxalyl chloride (0.44 ml) anddimethylformamide (1 drop) and stirred at ambient temperature for 3hours. Evaporation to dryness gave the acid chloride which was dissolvedin dichloromethane (150 ml) and treated with silver cyanate (1.0 g, 6.7mmoles), 2-(S)-2-dichloroacetoxymutilin 11-trifluoroacetate (2.3 g) andtriethylamine (0.65 ml) and stirred at ambient temperature for 18 hours.Filtration and evaporation of the filtrate to dryness followed bychromatography on silica gel, eluting with 25% ethyl acetate in hexanegave the title compound as a white foam (0.53 g, 15%).

[0202] (b) (6-tert-Butyloxycarbonylamino-3-pyridinylcarbonyl)carbamicAcid 2-(S)-hydroxymutilin 14-ester

[0203] (6-tert-Butyloxycarbonylamnino-3-pyridinylcarbonyl)carbamic acid2-(S)-2-dichloroacetoxy-mutilin 14-ester-11-trifluoroacetate (0.52 g) inabsolute ethanol (20 ml) was treated with 0.5N potassium hydroxide inethanol solution (2.5 ml, 1.2 mmoles) and stirred at ambient temperaturefor 4 hours. The mixture was evaporated to dryness and the residuepartitioned between water and ethyl acetate. The organics wereseparated, dried (Na₂SO₄) filtered and evaporated to dryness to give thetitle compound (0.37 g, 100%).

[0204] (c) (6-Amino-3-pyridinylcarbonyl)carbamic Acid2-(S)-hydroxymutilin 14-ester

[0205] (6-tert-Butyloxycarbonylamino-3-pyridinylcarbonyl)carbamic acid2-(S)-hydroxymutilin 14- ester(0.37 g) in dichloromethane (50 ml), wastreated with trifluoroacetic acid (2 ml) and stirred at ambienttemperature for 5 hours. The mixture was evaporated to dryness and theresidue partitioned between 10% potassium carbonate solution and 10%methanol/dichloromethane (2×100 ml). The organics were separated, dried(Na₂SO₄), filtered and evaporated to dryness. Chromatography on silicagel, eluting with 8% methanol/dichloromethane gave the title compound asa white solid (0.117 g, 37%). M.S. (−ve ion electrospray) m/z 498([M-H]⁻, 30%), 161 (100%).

EXAMPLES 2-27

[0206] (a) The following were prepared analogously to step (a) ofexample 1

Example % No. R yield Electrospray MS m/z 2

20 3

26 (−ve ion) 904 ([M − H]⁻, 100%) 4

54 (−ve ion) 904 ([M − H]⁻, 100%) 5

39 (−ve ion) 904 ([M − H]⁻, 100%) 6

44 (−ve ion) 904 ([M − H]⁻, 100%) 7

54 (−ve ion) 719 ([M − H]⁻, 100%) 8

40 (−ve ion) 810 ([M − H]⁻, 100%) 9

34 (−ve ion) 810 ([M − H]⁻, 100%) 10

12 11

62 12

71 (+ve ion) 902 (MNa⁺, 20%) 880(MH⁺, 20%)212(100%) 13

42 (−ve ion)720([M − H]⁻, 65%, 113(100%) 14

20 15

83 (−ve ion) 905 ([M − H]⁻, 40%), 113 (100%) 16

18.5 (−ve ion) 690 ([M − H]⁻, 90%), 123 (100%) 17

49 (−ve ion) 733 ([M − H]⁻, 100%) 18

18 (−ve ion) 819 ([M − H]⁻, 100%) 19

44 (−ve ion) 729 ([M − H]⁻, 100%) 20

15 (−ve ion) 934 ([M − H]⁻, 100%) 21

44 22

68 23

— 24

59 25

15 (+ve ion) 734 (MH⁺, 100%) 26

66 (−ve ion) 818 ([M − H]⁻, 100%) 27

76 28

29 (−ve ion) 819 ([M − H]⁻, 100%) 29

16 (−ve ion) 929 ([M − H]⁻, 100%)

[0207] 2-Aminopyrimidin-5-ylcarbonyl chloride hydrochloride for example10 was prepared by reflux of 2-aminopyrimidin-5-yl carboxylic acid (0.4g) (P. Schenone et. al., J. Heterocyclic Chem. 27 (1990)295) in thionylchloride (20 ml) for 4 hours followed by evaporation to dryness.

Example 3

[0208] (b) (5-Bis-t-butoxycarbonylaminonicotinoyl)carbamic acid2-(S)-hydroxymutilin 14-ester A solution of(5-bis-t-butoxycarbonylaminonicotinoyl)carbamic acid2-(S)-dichloroacetoxymutilin 14-ester-11-trifluororacetate (0.25 g) inethanol (25 ml) was treated with saturated aqueous NaHCO₃ (25 ml) andstirred vigorously for 2½ hours. The mixture was diluted with EtOAc (150ml) and water (150 ml), shaken and separated. The organic was dried andevaporated to give the title compound as a white solid (0.198 g). MS(−veion electrospray) m/z 698 ([M-H]⁻, 100%).

Examples 2, 4-17, 19-21 and 24-26

[0209] (b) The following were prepared analogously to step (b) of eitherExample 1 or Example 3.

Example % No. R yield Electrospray MS m/z 2

100 4

100 5

62 (−ve ion) 698 ([M − H]⁻, 100%) 6

76 (−ve ion) 698 ([M − H]⁻, 100%) 7

45 (−ve ion) 513 ([M − H]⁻, 100%) 8

97 (−ve ion) 604 ([M − H]⁻, 100%) 9

97 10

62 (−ve ion) 499 ([M − H]⁻, 100%) 11

100 12

99 (−ve ion) 672 ([M − H]⁻, 100%) 13

69 (−ve ion) 514 ([M − H]⁻, 100%) 14

18 (+ve ion) 991 (2MNa⁺, 100%), 485 (MH⁺, 40%) 15

18 (−ve ion) 699 ([M − H]⁻, 100%) 16

11 (−ve ion) 484 ([M − H]⁻, 60%), 122 (100%) 17

97 (−ve ion) 527 ([M − H]⁻, 100%) 19

70 (−ve ion) 523 ([M − H]⁻, 100%) 20

100 21

100 24

27 25

54 (+ve ion) 528 (MH⁺, 100%) 26

91 (−ve ion) 612 ([M − H]⁻, 100%)

Example 3

[0210] (c) (5-Aminonicotinoyl)carbamic acid 2-(S)hydroxymutilin 14-esterA solution of (5-bis-t-butoxycarbonylaminonicotinoyl)carbamic acid2-(S)-hydroxymutilin 14-ester (0.198 g) in trifluoroacetic acid (2 ml)was kept for 1 hour and evaporated. The residue was treated with EtOAc(10 ml) and saturated aqueous NaHCO₃ (10 ml), shaken and separated. Theorganic was dried and evaporated. Chromatography (EtOAc/MeOH) gave thetitle compound (0.084 g). MS (−ve ion electrospray) m/z 498 ([M-H]⁻,100%).

Examples 2, 4-6, 8-9, 11-12, 15, 20-21, 24 and 26

[0211] (c) The following were prepared analogously to step (c) of eitherexample 1 or example 3

Example % No. R yield Electrospray MS m/z 2

20 (−ve ion) 498 ([M − H]⁻, 38%), 268 (100%) 4

85 (−ve ion) 498 ([M − H]⁻, 100%) 5

77 (−ve ion) 558(MOAc⁻, 40%), 498([M − H]⁻, 85%), 162 (100%) 6

68 (−ve ion) 498 ([M − H]⁻, 70%), 162 (100%) 8

90 (−ve ion) 504 ([M − H]⁻, 30%), 168 (100%) 9

85 (−ve ion) 504 ([M − H]⁻, 10%), 168 (100%) 11

30 (−ve ion) 514 ([M − H]⁻, 55%), 178 (100%) 12

71 (−ve ion) 572 ([M − H]⁻, 100%) 15

61 (−ve ion) 499 ([M − H]⁻, 55%), 163 (100%) 20

49 (−ve ion) 528 ([M − H]⁻, 100%) 21

65 (−ve ion) 528 ([M − H]⁻, 100%) 24

94 (−ve ion) 499 ([M − H]⁻, 100%) 26

30 (−ve ion) 512 ([M − H]⁻, 100%)

Example 18 (2-N-methylaminopyrimidin-5-ylcarbonyl)carbamic acid2-(S)-hydroxymutilin 14-ester

[0212] (b) (2-N-methylaminopyrimidin-5-ylcarbonyl)carbamic Acid2-(S)-dichloroacetoxy-11-O-trifluoroacetylmutilin 14-ester

[0213] BOC-protected material from step (a) (see table) was deprotectedwith TFA using the procedure of Example 3, step (c) (100%). MS (−ve ionelectrospray) m/z 719 ([M-H]⁻, 100%)0

[0214] (c) (2-N-methylaminopyrimidin-5-ylcarbonyl)carbamic Acid2-(S)-hydroxymutilin 14-ester

[0215] Material from step (b) was treated according to the procedure ofExample 3, step (b) to give the title compound (64%). MS (+ve ionelectrospray) m/z 515 (MH⁺, 100%)

Example 22(b) (6-Amino-5-nitronicotinoyl)carbamic Acid2-(S)-hydroxymutilin 14-ester

[0216]

[0217] (6-Bis-t-butoxycarbonylamino-5-nitronicotinoyl)carbamic acid2-(S)-dichloroacetoxy-11-O-trifluoroacetylmutilin (see table) wastreated with TFA according to Example 3, step (c) followed by baseaccording to Example 3, step (b) to give the title compound (95%), MS(−ve ion chemical ionisation) m/z 543 ([M-H]⁻, 100%)

Example 23(b) (2-Amino-6-methoxypyrimidin-4-ylcarbonyl)carbamic Acid2-(S)-hydroxymutilin 14-ester

[0218]

[0219](2-Bis-t-butoxycarbonylamino-6-methoxypyrimidin-4-ylcarbonyl)carbamicacid 2-(S)-dichloroacetoxy-11-O-trifluoroacetylmutilin (see table) wastreated with TFA according to Example 3, step (c) followed by baseaccording to Example 3, step (b) to give the title compound. MS (−ve ionelectrospray) m/z 529 ([M-H]⁻, 60%), 193 (100%).

Example 27 (3-Amino-6-pyridazinylcarbonyl)carbamic Acid2-(S)-hydroxymutilin 14-ester hydrochloride

[0220]

[0221] (b) (Tetrazolo [1,5-b] pyridazin-6-ylcarbonylcarbamic Acid(2S)-2-dichloroacetoxy-11-O-trifluoroacetylmutilin 14-ester

[0222] The title compound was prepared from1-(3-chloro-6-pyridazinylcarbonyl)carbamic acid(2S)-2-dichloroacetoxy-11-O-trifluoroacetyl mutilin 14-ester (see table)(1.5 g) by treatment with sodium azide (0.162 g) in DMF (20 ml) atambient temperature for 4 hours. The mixture was then evaporated todryness and the residue extracted with ethyl acetate (50 ml) and washedwith water (3×50 ml), dried and evaporated to give (1.02 g, 70%). M.S.(−ve ion electrospray) m/z 731 ([M-H]⁻, 15%), 164 (100%).

[0223] (c) (3-Triphenylphosphoranylideneamino-6-pyridazinylcarbonyl)carbamic Acid(2S)-2-dichloroacetoxy-11-O-trifluoroacetylmutilin 14-ester.

[0224] (Tetrazolo [1,5-b] pyridazin-6-ylcarbonyl)carbamicacid-(2S)-2-dichloroacetoxy-11-O-trifluoroacetylmutilin 14-ester (0.45g) was heated in chlorobenzene (10 ml) with triphenyl-phosphine (0.165g) at 110° C. for 18 hours. Evaporation followed by chromatography onsilica gel eluting with 50% ethyl acetate in hexane gave the titlecompound (0.255 g, 43%). M.S. (+ve ion electrospray) m/z 967 (MH⁺, 80%),839 (100%).

[0225] (d) (3-Amino-6-pyridazinylcarbonyl)carbamicAcid-(2S)-2-dichloroacetoxy-11-O-trifluoroacetylmutilin 14-ester

[0226] (3-Triphenylphosphoranylideneamino-6-pyridazinylcarbonyl)carbamicacid-(2S)-2-dichloroacetoxy-11-O-trifluoroacetylmutilin 14-ester (0.25g) was treated with glacial acetic acid (5 ml) and water (0.5 ml) andheated at 100° C. for 1 hour. The mixture was evaporated to dryness andthe residue extracted with ethyl acetate and washed with saturatedaqueous sodium bicarbonate solution, dried and evaporated to dryness togive the title compound as a 1:1 mixture with triphenylphosphine oxide(0.23 g, 88%). M.S (−ve ion electrospray) m/z 705 ([M-H]⁻, 18%), 375(100%).

[0227] (e) (3-Amino-6-pyridazinylcarbonyl)carbamicAcid-(2S)-2-hydroxymutilin 14-ester Hydrochloride

[0228] (3-Amino-6-pyridazinylcarbonyl)carbamicacid-(2S)-2-dichloroacetoxy-11-O-trifluoro acetyl mutilin 14-ester (0.23g) was treated with aqueous sodium bicarbonate as in Example 3, step (b)then treated with ethereal hydrogen chloride to give the title compound(0.05 g, 41%). M.S. (−ve ion electrospray) m/z 499 ([M-H]⁻, 100%).

EXAMPLE 28 (3-N-methylpyridazin-6-ylcarbonyl)carbamic Acid2-(S)-hydroxymutilin 14-ester

[0229] (b) (3-N-methylpyridazin-6-ylcarbonyl) carbamic Acid2-(S)-dichloroacetoxy-11-O-trifluoroacetylmutilin 14-ester

[0230] BOC-protected material from step (a) (see table) was deprotectedwith TFA using the procedure of Example 3, step (c) (73%). MS (−ve ionelectrospray) m/z 720 ([M-H]⁻, 100%)

[0231] (c) (3-N-methylpyridazin-6-ylcarbonyl)carbamic Acid2-(S)-hydroxymutilin 14-ester

[0232] Material from step (b) was treated according to the procedure ofExample 3, step (b) to give the title compound (44%). MS (−ve ionelectrospray) m/z 513 ([M-H]⁻, 100%).

EXAMPLE 29 (6-Amino-5-cyanonicotinoyl)carbamic acid 2-(S)-hydroxymutilin14-ester

[0233] (b) (6-Amino-5-cyanonicotinoyl)carbamic Acid2-(S)-dichloroacetoxy-11-O-trifluoroacetylmutilin 14-ester BOC-protectedmaterial from step (a) (see table) was deprotected with TFA using theprocedure of Example 3, step (c) (76%). MS (−ve ion electrospray) m/z729 ([M-H]⁻, 100%).

[0234] (c) (6-Amino-5-cyanonicotinoyl)carbamic acid 2-(S)-hydroxymutilin14-ester Material from step (b) was treated according to the preocedureof Example 3, step (b) to give the title compound (60%). MS (−ve ionelectrospray) m/z 523 ([M-H]⁻, 100%).

EXAMPLE 30 [2-(1-Carboxamidomethylpiperidin-4-yl)thiazole-4-carbonyl]carbamic Acid 2-(S)-hydroxymutilin 14-ester

[0235]

[0236] A solution of [2-(piperidin-4-yl)thiazole-4-carbonyl]carbamicacid 2-(S)-hydroxymutilin 14-ester (example 12, 120 mg) in acetonitrile(3.5 ml)/DMF (0.5 ml) was treated with potassium carbonate (73 mg) and2-bromoacetamide (29 mg) and stirred overnight. The mixture was dilutedwith EtOAc (10 ml), washed with water (3×10 ml), dried and evaporated.Chromatography, eluting with chloroform/methanol/0.88NH₃ (aq) 94:6:0.6gave the title compound (90 mg). MS (+ve ion electrospray)m/z 631 (MH⁺,30%), 269 (100%).

EXAMPLE 31 [2-(1-Cyanomethylpiperidin-4-yl)thiazole-4-carbonyl]carbamicAcid 2-(S)-hydroxymutilin 14-ester

[0237] Using bromacetonitrile as alkylating agent, an analogous reactionto that of example 30 gave the title compound (74%) MS(−ve ionelectrospray) m/z 611 ([M-H]⁻, 100%).

EXAMPLE 32 (6-aminopyridin-2-ylcarbonyl)carbamic acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester

[0238]

[0239] A solution of (6-aminopyridin-2-ylcarbonyl)carbamic acid2-(S)-hydroxymutilin 14-ester (Example 4) (150 mg) in ethanol (20 ml)was treated with 10% Pd/C (50 mg) and stirred under hydrogen atatmospheric pressure overnight. The catalyst was filtered off and thefiltrate evaporated to give the title compound (130 mg). MS (+ve ionelectrospray)m/z 502 (, 40%), 524 (MNa⁺, 65%), 565 (100%).

EXAMPLE 33 (6-Amino-5-cyanonicotinoyl)carbamic Acid19,20-dihydro-2-(S-hydroxymutilin 14-ester

[0240] (6-Amino-5-cyanonicotinoyl)carbamic acid 2-(S)-hydroxymutilin14-ester was hydrogenated according to the procedure of example 32 (butusing dioxan as solvent instead of EtOH) to give title compound (62%).MS (−ve ion electrospray) m/z 525 ([M-H]⁻, 100%).

EXAMPLE 34 (3-Oxo-3,4-dihydropyrido[2,3-b]pyrazin-7-ylcarbonyl)carbamicAcid 19,20-dihydro-2-(S)-hydroxymutilin 14-ester

[0241]

[0242] (a) (5,6-Diaminonicotinoyl)carbamic Acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester

[0243] (6-Amino-5-nitronicotinoyl)carbamic acid 2-(S)-hydroxymutilin14-ester (Example 22) was hydrogenated according to the procedure ofExample 32 to give the title compound (86%). MS (+ve ion chemicalionisation) m/z 517 (MH⁺, 100%).

[0244] (b) (3-Oxo-3,4-dihydropyrido[2,3-b]pyrazin-7-ylcarbonyl)carbamicAcid 19,20-dihydro-2-(S)-hydroxymutilin 14-ester

[0245] A solution of (5,6-diaminonicotinoyl)carbamic acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester (118 mg) in ethanol (10 ml)was treated with a solution of ethylglyoxylate (150 ml of 4.9 M toluenesolution) and heated to 50° C. for 3 hours. Solvent was evaporated andthe residue chromatographed, eluting with dichloromethane/methanol 97:3to give the title compound (13 mg). MS (+ve ion chemical ionisation) m/z555 (MH⁺, 100%).

EXAMPLE 35 (2-Aminothiazol-5-ylcarbonyl)carbamic Acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester

[0246]

[0247] (a) (2-t-Butoxycarbonylaminothiazol-5-ylcarbonyl)carbamic acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester(2-t-Butoxycarbonylaminothiazol-5-ylcarbonyl)carbamicacid-2-(S)-hydroxymutilin 14-ester (example 8, step (b))) washydrogenated as described in Example 32 to give the title compound(46%). MS (−ve ion electrospray) m/z 606 ([M-H]⁻, 50%/), 268 (100%).

[0248] (b)) (2-Aminothiazol-5-ylcarbonyl)carbamic acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester BOC-protected compound fromstep (a) was deprotected as described in Example 3 step (c) to give thetitle compound (46%). MS (−ve ion electrospray) m/z 506 ([M-H]⁻, 100%).

EXAMPLE 36 (5-Amino-6-methoxynicotinoyl)carbamic Acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester

[0249]

[0250] (5-Amino-6-methoxynicotinoyl)carbamic acid 2-(S)-hydroxymutilin14-ester was hydrogenated as described in example 32 to give the titlecompound. MS (−ve ion electrospray) m/z 530 ([M-H]⁻, 50%), 192 (100%).

EXAMPLES 37-39

[0251] (a) The following were prepared analogously to step (a) ofExample 1, using2-(S)-2-dichloroacetoxy-19,20-dihydro-11-O-tifluoroacetylmutilin(Preparation 14).

Example % No R yield Electrospray MS m/z 37

18 (−ve ion) 821 ([M − H]⁻, 100%) 38

28 (−ve ion) 722 ([M − H]⁻, 100%) 39

16

Example 37 (b) (2-Methylaminopyrimidin-5-ylcarbonyl)carbamic Acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester

[0252]

[0253](2-N-t-butoxycarbonyl-N-methylaminopyrimidin-5-ylcarbonyl)carbamic acid2-(S)-dichloracetoxy-19,20-dihydro-11-O-trifluoroacetylmutilin 14-ester(see table) was treated with TFA according to the procedure of Example3, (step (c) (100%). [MS (−ve ion electrospray) m/z 721 ([M-H]⁻, 100%)]and then with base according to the procedure of Example 3, step (b)(44%). MS (−ve ion electrospray) m/z 515 ([M-H]⁻, 100%)

Example 38 (b) (2-Methoxypyrimidin-5-ylcarbonyl)carbamic Acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester

[0254]

[0255] (2-Methoxypyrimidin-5-ylcarbonyl)carbamic acid2-(S)-dichloroacetoxy-19,20-dihydro-11-O-trifluoroacetylmutilin 14-esterwas deprotected according to the procedure of Example 3, step (b) toprovide the title compound (43%). MS (+ve ion electrospray) 518 (MH⁺,100%).

Example 39 (b) (6-Aminonicotinoyl)carbamic Acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester

[0256]

[0257] (6-t-Butoxycarbonylaminonicotinoyl)carbamic acid2-(S)-dichloroacetoxy-19,20-dihydro-11-O-trifluoroacetylmutilin 14-ester(see table) was deprotected according to the procedure of Example 3,step (b) (65%) [MS (−ve ion chemical ionisation) m/z 600 ([M-H]⁻, 100%)]and then according to Example 3, step (c) (39%). MS (−ve ionelectrospray) m/z 500 ([M-H]⁻, 100%).

[0258] Biological Data

[0259] Compounds of the present invention were assessed foranti-bacterial activity in a conventional MIC assay against a range ofpathogenic organisms.

[0260] Examples 1 to 39 were found to have MICs ≦4 μg/ml againstStaphylococcus aureus Oxford, Streptococcus pneumoniae 1629, Moraxellacatarrhalis Ravasio, and Haemophilius influenzae Q1.

[0261] The improved stability of the 2S-hydroxy compounds wasdemonstrated using human liver microsome preparations. Thus, for thecompounds in which R¹=2-amino-4-pyridyl and R²=vinyl, the intrinsicclearances (CLi, a measure of rate of metabolism) in the presence ofhuman liver microsomes were found to be: 2α-H, CLi>50 ml/min/g liver;2α-OH, CLi=6.5 ml/min/g liver.

1. A compound of Formula (I):

in which: R¹ is a 5- or 6-membered optionally substituted heteroarylgroup; and R² is vinyl or ethyl.
 2. A compound as claimed in claim 1 inwhich R¹ comprises a 5 or 6-membered single ring comprising 1 or 2nitrogen atoms and optionally comprising a further heteroatom selectedfrom oxygen or sulphur; or a 5 or 6-membered ring comprising 3 nitrogenatoms; or a 5 or 6-membered ring comprising 1 or 2 nitrogen atoms fusedto a benzene ring or a second 5 or 6-membered optionally substitutedheteroaryl ring comprising 1 or 2 nitrogen atoms.
 3. A compound asclaimed in claim 1 or 2 in which R¹ comprises pyridine, pyridazine,pyrimidine, pyrazine, isoxazole, thiazole, imidazole, pyrazole,1,2,3-triazole, 1,2,4-triazole, benzimidazole,3-oxo-3,4-dihydropyrido[2,3-b]pyrazine, or pyrazolo[1,5-a]pyrimidine. 4.A compound of formula (I) as claimed in any one of claims 1 to 3 inwhich R¹ comprises pyridine, pyrimidine, and thiazole.
 5. A compound offormula (I) as claimed in any one of claims 1 to 4 in which asubstituent for R¹ is selected from amino, mono- or di-(C₁₋₆)alkylamino,(C₁₋₆)alkyl, (C₁₋₆)alkoxy, nitro and N-containing heterocyclyl.
 6. Acompound of formula (I) as defined in claim 1 selected from the groupconsisting of: (6-Amino-3-pyridinylcarbonyl)carbamic acid2-(S)-hydroxymutilin 14-ester; (5-Aminonicotinoyl)carbamic acid2-(S)-hydroxymutilin 14-ester;(2-N-methylaminopyrimidin-5-ylcarbonyl)carbamic acid2-(S)-hydroxymutilin 14-ester; (3-Amino-6-pyridazinylcarbonyl)carbamicacid 2-(S)-hydroxymutilin 14-ester;(3-N-methylpyridazin-6-ylcarbonyl)carbamic acid 2-(S)-hydroxymutilin14-ester; (6-Amino-5-cyanonicotinoyl)carbamic acid 2-(S)-hydroxymutilin14-ester; [2-(1-Carboxamidomethylpiperidin-4-yl)thiazole-4-carbonyl]carbamic acid 2-(S)-hydroxymutilin 14-ester;[2-(1-Cyanomethylpiperidin-4-yl)thiazole-4-carbonyl]carbamic acid2-(S)-hydroxymutilin 14-ester; (6-aminopyridin-2-ylcarbonyl)carbamicacid 19,20-dihydro-2-(S)-hydroxymutilin 14-ester;(6-Amino-5-cyanonicotinoyl)carbamic acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester;(3-Oxo-3,4-dihydropyrido[2,3-b]pyrazin-7-ylcarbonyl)carbamic acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester;(2-Aminothiazol-5-ylcarbonyl)carbamic acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester;(5-Amino-6-methoxynicotinoyl)carbamic acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester;(6-Amino-5-nitronicotinoyl)carbamic acid 2-(S)-hydroxymutilin 14-ester;(2-Amino-6-methoxypyrimidin-4-ylcarbonyl)carbamic acid2-(S)-hydroxymutilin 14-ester; and a compound of formula (I) in which R²is ethyl and R¹ is selected from:


7. A compound of formula (I) as defined in claim 1 selected from thegroup consisting of: (5-Amino-6-methoxy-3-pyridinylcarbonyl)carbamicacid 2-(S)-hydroxymutilin 14-ester,(5-Amino-6-methoxy-3-pyridinylcarbonyl)carbamic acid19,20-dihydro-2-(S)hydroxymutilin 14-ester;(6-Amino-3-pyridinylcarbonyl)carbamic acid19,20-dihydro-2-(S)-hydroxymutilin 14-ester;(6-Dimethylamino-3-pyridinylcarbonyl)carbamic acid 2-(S)-hydroxymutilin14-ester; and (3-Amino-6-pyridazinylcarbonyl)carbamic acid2-(S)-hydroxymutilin 14-ester.
 8. A pharmaceutical compositioncomprising a compound of formula (I) as claimed in claim 1 and apharmaceutically acceptable carrier or excipient.
 9. A compound offormula (I) as claimed in claim 1 for use in therapy.
 10. A process forpreparing a compound of formula (I) as claimed in claim 1 which processcomprises reacting a compound of formula (II):

in which X and P are hydrogen or a hydroxyl protecting group, such as anacyl group, and R² is as defined in claim 1; with an acyl isocyanate offormula R^(1A)CONCO in which R^(1A) is R¹ as hereinbefore defined or agroup convertible into R¹, for instance a group comprising a protectedsubstituent therein and thereafter and if necessary: (i) deprotecting agroup P and/or X to generate a hydroxyl group at position 2 or 11,respectively, (j) converting a group R^(1A) to R¹, for instance removinga protecting group, (k) converting a group R¹ to another group R¹, and(I) hydrogenating the vinyl group at position 12 to form an ethyl group.11. A compound of formula (II)

in which P and X are hydroxyl protecting groups, and R² is as defined inclaim 1.